in the liver (Mitchell, Neumann and Draper, i959). Cameron (1964) using in vitro liver infusion techniques observed that 90% of the radioactivity of estrone-14C ...
LIVER AND GASTROINTESTINAL OF DIETHYLSTILBESTROL T. L.
HUBER,
IN
METABOLISM S H E E P 1. 2
G. W. HORN AND R. E. BEADLE
University o] Georgia, Athens 30601 I E T H Y L S T I L B E S T R O L (DES) has been D used as a growth stimulant in ruminants for many years and is administered orally and by subcutaneous implantation. That much larger quantities are required when DES is administered orally suggests the occurrence of large losses of DES before reaching its site of action. Gastrointestinal absorption data are not available; however, it has been shown that conjugation with glucuronic acid does occur in the liver (Mitchell, Neumann and Draper, i959). Cameron (1964) using in vitro liver infusion techniques observed that 90% of the radioactivity of estrone-14C was removed from blood after a single passage through the human liver. Furthermore, Hartiala (1961) using preparations of duodenum from the dog has shown the occurrence of glucuronide synthesis when steroid estrogen or stilbestrol were used as substrate. Thus, should the site of the anabolic action of DES in ruminants be extrahepatic, extensive gastrointestinal and liver conjugation of orally administered DES would allow only small quantities of free DES to reach these tissues. The purpose of the present study was to estimate the quantity of 1*C-DES removed from blood during a single passage through perfused sheep liver, and to determine the forms of DES in blood after one liver passage and after a 1-hr. perfusion period. Also, gastrointestinal tissues were evaluated for glucuronide synthesis using DES as substrate. Materials and M e t h o d s Five adult sheep, three wethers and two ewes, were used in the experiments. Following a 24 hr. fast the sheep were anesthesized with sodium pentobarbital and bled from the carotid artery. The blood was immediately defibrinated, by shaking with glass beads and by stirring with a glass rod, and filtered through four layers of cheesecloth. When Published as Paper No. 881, University of Georgia Institute of Comparative Medicine, Department of Physiology and Pharmacology. 2 This project supported in part by Lilly Research Laboratories grant.
carotid flow had decreased to a minimum the abdominal cavity was entered through an incision caudal to the last right rib extending from the dorsal vertebra to the xiphoid cartilage. The liver was freed of all attachments with the portal vein, hepatic artery and posterior vena cava attachments at the diaphragm being the last severed. For the gastrointestinal studies, the caudal 15 cm of the duodenum was removed and placed in 37C Krebs-Ringer bicarbonate buffer. Liver Per/usion. The perfusion chamber is shown in figure 1. I t was constructed with 1.9 cm plywood and had an inside dimension of 55 x 55 x 190 centimeters. Glass doors were mounted on one side for entry and visual observation during perfusion. A uniform temperature of 37 C was maintained in all areas of the chamber by a thermostat-controlled heater and fan. The livers were placed on a plastic-coated screen located near the top of a 26.3 cm plastic funnel and residual blood was washed from the livers by allowing warm Krebs-Ringer bicarbonate buffer to flow through a catheter placed in the portal vein. The hepatic artery and bile duct were then catheterized. The portal vein catheter was connected to a reservoir located above the liver, the hepatic arterial catheter connected to a pulsatile blood pump and the perfusions were begun. After the livers were removed from the sheep, approximately 5 to 10 min. were required to establish in vitro circulation. Efferent blood flowed from the post vena cava into the plastic funnel, a separatory funnel and oxygenator. The separatory funnel was calibrated so that flow rate could be determined by closing the funnel stopcock. As the blood passed through the oxygenator it was arterialized by a mixture of 95% 02 and 5% CO~. which was humidified by bubbling through two tubes of 37C water. The arterialized blood was pumped to the portal reservoir by a peristaltic pump and to the hepatic artery by the pulsatile blood pump. All catheters and tubing were silastic. 3 9 s Dow Corning, Midland, Mich.
786 JOURNAL OF ANIMAL SCIENCE, vo]. 34, tin. 5, 1972
DIETHYLSTILBESTROL METABOLISM
Figure 1. Perfusion chamber with doors open. A----Portal reservoir, B----Portal vein catheter, C--~Hepatic artery catheter, D=Plastic funnel conntaining liver, E=Separatory funnel, F = Oxygenator, G=Water tubes, H=laeristaltic pump, I--~Pulsatile pump, J~-Bile duct catheter. When a steady rate of perfusion was obtained (approximately 20 rain.) 20 ~c of DES monoethyl_l_14C 4 in 40% ethanol were added to approximately 300 ml of blood and placed in the portal reservoir and allowed to flow through the livers. The blood was followed by 200 ml of Krebs-Ringer bicarbonate buffer to insure that all blood had passed through the livers. The blood and buffer were collected, mixed, measured, and an aliquot taken for subsequent analysis. The mixture was then added back to the perfusion system and the perfusions were continued for 1 hour. At the conclusion of the perfusion period the livers, catheters, and tubing were washed free of Amersharn/Searle, Arlington Heights, Ill.
787
perfusate with buffer. The perfusate aliquots and livers were frozen until analyzed. Gastrointestinal Studies. The duodenum was everted and washed with Krebs-Ringer buffer. Three 2.5 cm sacs were prepared by ligating one end of the everted duodenum with a nylon ligature, filling with 0.5 ml of Krebs-Ringer bicarbonate buffer containing glucose and sodium lactate in concentrations of 200 rag/100 ml, and closing the open encl with ligature. The sacs were incubated in 10 ml of the above solution plus 0.2 ~c of the 14C-DES for 90 min. at 37 C under an atmosphere of 95% 02 and 5% CO2. At the conclusion of the incubation period the sacs were washed with buffer and the contents of the three sacs combined for analysis. Approximately 100 mg of rumen mucosa were incubated under the conditions described for the intestinal sacs. Triplicate incubations were conducted and following the incubation period the incubation mixtures were contrifuged and the supernatants were combined for analysis. In Vivo Conjugate Studies. An adult sheep was anesthetized with sodium pentobarbital and the anterior mesenteric artery and portal vein were catheterized. An aqueous solution of DES-glucuronide was injected into the anterior mesenteric artery and a 2-rain. continuous blood sample was collected from the portal vein. Analytical. The extraction procedure described by Teague and Brown (1951) was used to partition the radioactivity of blood, bile, incubation solutions, and liver into free phenolic and conjugate fractions. Samples were taken from peripheral and central areas of the livers and prepared for extraction by homogenizing approximately 1 g of liver in 20 ml of distilled water. Aliquots of the ether (free phenolic) and water (conjugate) extracts were added to a scintillator solution composed of ethanol, toluene, 2,5-diphenvloxazole (PPO) and 5Dhenoloxazole (POPOP) and counted in a Packard Tri-Carb Scintillation Spectrometer. Extraction efficiency ,was determined by chromatographing aliqu0ts of the extracts against D~re free DES on Whatman :~ 1 paper usine 60% benzene and 40% ethanol as the mobile phase. Since liver was the only tissue involved in the perfusion experiments, it would be expected that conjt~gates in the water extracts of bile would be the same as those in the blood and liver. Thus, bile was used to characterize the conjugate formed in the perfusion experiments. One milliliter of bile was
788
HUBER, HORN AND BEADLE
diluted with 20 ml of water, acidified to pH 4.0 with 0.1 N HC1 and extracted three times with 2.5 volumes of diethyl ether. The ether extract was reduced to dryness (40 C, flash evaporator) and dissolved in 10 ml of distilled water. Further purification of the extract was done by a nonionic absorption method described by Robbins, Hedde and Bakke (1969). The purified bile extract was then chromatographed by gel filtration on a column (1.0 x 100 cm) of Sephadex LH-20 developed in methanol. The 14C fraction was then chromatographed on Whatman ~ 1 paper strips (19 x 55 cm) and developed in butanol, ammonium hydroxide and water (4:1:5). Homogeneity of the radioactive fraction was further evaluated by thin-layer chromatography on silica gel-HF plates developed in butanol, acetic acid and water (4:1:5). To further characterize the conjugate fraction, a 1 ml adliquot of the water extract of bile was subjected to hydrolysis with 300 mg of fl-glucuronidase (Worthington Biochem. Corp.). After a 24-hr. incubation period, the incubation mixture was extracted as described above and aliquots of the extracts chromatographed. Results and Discussion After a steady rate of perfusion was obtained, mean hepatic venous flow was 256 ml/min/100 g of liver. Schambye (1955) estimated mean portal flow in sheep to be 266 mlJmin/100 g of liver. The contribution of the hepatic artery to total hepatic venous flow has been estimated to be 20% by Katz and Bergman ( 1969). Applying this estimate to the present data, a portal flow of approximately 235 ml/min/100 g liver may be calculated. This value is well within the range of 185 to 299 m l / m i n / l O 0 g of liver reported by Schambye. In some experiments, Po2 and Pco2 of the blood leaving the liver (venous) and of blood leaving the oxygenator (arteriolized) were determined. Typical values for Po~ were 89 and 102 mm Hg and for Pco2, 24 and 18 mm Hg for venous and arteriolized blood, respectively. These data indicate that the livers were adequately oxygenated and that oxidative processes were functional. When the ether fractions were chromatographed the radioactivity was found to have an Rf value identical to that obtained when pure free DES was chromatographed. When the water fractions were chromatographed,
the radioactivity remained at the point of origin. These observations indicate that the radioactivity prior to extraction was distributed between at least two compounds and that the radioactivity in the ether fraction represented free DES. In characterizing the radioactivity in the water fraction of bile, recovery of radioactivity was quantitative through the cleanup steps using the partitioning nonionic adsorption and gel filtration techniques. The radioactivity chromatographed as a single band in the 120 to 140 ml eluate from the LH-20 column. Gel filtration on LH-20 has been used to separate sulfate and glucuronide conjugates excreted in chicken urine (Paulson and Zehr, 1971) and of conjugates in rat and goat urine (Robbins, Bakke and Fell, 1970; Robbins, et al., 1969). Further proof of a single metabolite was obtained by paper and thin-layer chromatography. The metabolite gave a single band with paper ( R f = 0 . 5 5 ) and thin-layer chromatography ( R f = 0 . 6 9 ) . These data indicate that DES was conjugated by the liver to a single, watersoluble metabolite. When this metabolite was subjected to enzymatic hydrolysis by fl-glucuronidase and the incubation mixture rechromatographed, all the radioactivity had an Rf equal to free DES. Thus all evidence obtained by chromatography and enzymatic hydrolysis indicates that DES is conjugated to only the glucuronide by sheep liver. Hinds et al. (1965) have reported similar observations. Although the radioactivity in the water fractions of the gastrointestinal studies was not characterized, one would expect the presence of the glucuronide since it has been the only conjugate detected in urine of sheep and cattle. The liver perfusion data are presented in table 1. Mean retention of radioactivity by the livers after a single passage of 14C-DES through the livers was 76.1%. When expressed on a per gram of liver basis, the mean retention percentage was 0.21%. Of the total radioactivity remaining in the perfusate after the initial passage, 38.5% was associated with free DES and 61.5% was associated with the glucuronide. These data indicate that DES conjugation with glucuronic acid is a very rapid process. Diczfalusy, Franksson and Martinsen (1961) made similar observations by injecting 17-fl-estradiol into the arterial blood of a human intestinal loop and detectin~ the glucuronide in the efferent blood. The rapidity of conjugation suggests that DES is not transformed to other metabolites prior to conjugation.
D I E T H Y L S T I L B E S T R O L METABOLISM TABLE
1. L I V E R
PERFUSION
Perfusate
789
DATA ~ Liver 1 hr. ~
Radioactivity retained % of t o t a l / l i v e r % of t o t a l / g of l i v e r Composition Free DES DES-glucuronide R a d i o a c t i v i t y i n bile a
IX b
1 hr. c
....... .......
....... .......
38.5+11.60 6 1 . 5 • 11.60 .......
IX b
76.1• 0.2+0.01
Central
Peripheral
Bile
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
0.7____+0.54 ....... 4.7• 5.3• 99.3___+0.54 ....... 95.3~1.54 94.7• . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3• 97.6+0.64 6.1--+~3.59
a Mean Percentages+SEM; n z 5 . b Single passage of perfusate through liver. e At end of the 1-hr. perfusion period. a Percent of x4C-DES incorporated into bile during the 1-hr. perfusion period.
From the present data, one can calculate that from an oral 2 mg dose of DES given to sheep, assuming all was absorbed and reached the liver in the free form, only about 184 /~g of free DES would be available for systemic distribution per day. Considerable fl-glucuronidase activity has been reported in intestinal tissue of sheep (Kellie, 1961). However, in the current study no evidence of hydrolysis was found when the glucuronide was injected into the anterior mesenteric artery, thus indicating that additional free DES would not be made available by hydrolysis of the glucuronide. However, definite conclusions cannot be made from these data since quantitative recovery of radioactivity was not made and portal blood was sampled for only a very short period. It may be possible that enzymatic hydrolysis of the glucuronide by tissues is accomplished over a longer period of time. No information is available regarding the anabolic activity of DES-glucuronide but Simpson and Smith (1948) have observed a large reduction in .the estrogenic activity following conjugation. Although data are not available concerning absorption rates of DES from 3 mg, implanted DES pellets available for sheep, Hale et al. (1959) have estimated the absorption rate from 12 mg pellets in cattle. These workers estimated that approximately 56 to 74 ~g of DES were absorbed per day from 12 mg DES pellets. Since the liver receives approximately 30% of the cardiac output, the quantity of pellet absorbed DES distributed to extrahepatic tissues would be approximately 39 to 52 t~g per day. A comparison of the calculated quantity of free DES from a 12 mg pellet and of that calculated from a 2 mg dose delivered directly to the liver reaching the systemic circulation suggests that considerable losses of orally administered free DES occur
prior to the liver. The percentage of orally administered DES absorbed is not known; however, results of the gastrointestinal studies show that rumen mucosa can form DESglucuronide and that intestine can form and transport DES-glucuronide. The mean ratio of radioactivity appearing in the free phenolic and conjugate fractions of the rumen mucosa incubation mixture was 5 to 1 and the ratio appearing on the serosal side of the intestinal sacs was 6.1 to 1. If absorption loss and gastrointestinal conjugation were quantitated, the quantity of free DES reaching extrahepatic tissues might be near that calculated from implanted pellets. Following the 1 hr. perfusion period 99.3 % of the perfusate radioactivity was found in the conjugate fraction. The perfusate also contained approximately 50% more radioactivity indicating that a portion of the initial retained radioactivity was released from the liver. This was not totally unexpected since other workers have found radioactivity in the urine for several hours following the injection of labelled DES. At the conclusion of the experiments the conjugate fraction of the central liver samples and peripheral liver samples contained 95.3% and 94.7% of the radioactivity, respectively. The percentage of radioactivity in bile appearing in the conjugate fraction was 97.6%. Taking into account the radioactivity in the aliquot of perfusate removed after initial passage through the livers, the mean percentage of radioactivity recovered in bile was 6.1%. It would appear that biliary excretion may involve two separate mechanisms, conjugation and excretion. The increase in radioactivity of the conjugate fraction of the perfusate during the 1-hr. perfusion period suggests that the secretory mechanism may be a rate-limiting process.
HUBER, HORN AND BEADLE
790 Summary
The metabolism of diethylstilbestrol ( D E S ) monoethyl-l-14C b y liver and gastrointestinal tissue was studied in five mature sheep. In vitro liver perfusions, everted intestinal sacs, and rumen mucosa incubation procedures were employed. Mean retention of radioactivity b y the livers after a single passage of 14C-DES through the livers was 76.1%. Of the total radioactivity remaining in the perfusate after the initial passage, 38.5% was free DES and 61.5% was the glucuronide. Following a 1-hr. perfusion period 99.3 % of the perfusate radioactivity was in the conjugate fraction and the perfusate contained approximately 50% more radioactivity indicating release of initially retained activity. Approximately 95.0% of the radioactivity present in the livers after the 1-hr. perfusion period was in the conjugate fraction. I t was observed that rumen mucosa can form DES-glucuronide and that intestine can form and transport DES-glucuronide. Evidence for tissue hvdrolysis of the glucuronide was not obtained.
Literature Cited Cameron, C. B. 1964. The Liver II. Academic Press, New York. Diczfalusy, E., C. Franksson and B. Martinsen. 1961. Oestrogen conjugation by the human intestinal tract. Acta Endocrinol. 38:59. Hate, W. H., W. C. Sherman, E. A. White, G. Kuhn,
R. B. Schnell, W. M. Reynolds and H. G. Luther. 1959. Absorption of diethylsfilbestrol pellets in steers. J. Anita. Sci. 18:1201. Hartiala, K. 1961. Experimental studies of gastrointestinal conjugation functions. Biochem. Pharm. 6:82. Hinds, F. C., H. H. Draper, G. E. Mitchell, Jr. and A. L. Neumann. 1965. Metabolism of labelled diethytstilbestrol in ruminants. J. Agr. Food Chem. 13:257. Katz, M. L. and E. N. Bergman. 1969. Simultaneous measurements of hepatic and portal venous blood flow in the sheep and dog. Amer. J. Physiol. 216: 946. Kellie, A. E. 1961. The metabolism of steroid conjugates: Androstenolone sulphate and glucuronoside. J. Endocrinol. 22 :p-i. Mitchell, G. E. Jr., A. L. Neumann and H. H. Draper. 1959. Metabolism of tritium labelled diethylstilbestrol by steers. J. Agr. Food Chem. 7:509. Paulson, G. D. and M. V. Zehr. 1971. Metabolism of p-chlorophenyl N-methylcarbamate in the chicken. J. Agr. Food Chem. 19:471. Robbins, J. D., J. E. Bakke and V. J. Fell. 1970. Metabolism of benzo [b] thien-4-yl methylcarbamate (Mobam) in dairy goats and a lactating cow. J. Agr. Food Chem. 18:130. Robbins, J. D., R. D. Hedde and J. E. Bakke. 1969. Extraction of conjugates from urine by nonionic adsorption. Separation Sci. 4:345. Schambye, P. 1955. Experimental estimation of the portal vein blood flow in sheep. Nord. Vet.-Med. 7:1001. Simpson, S. A. and A. E. Wilder Smith. 1948. The isolation and properties of the monoglucuronides of stilbestrol, hexoestrol and dienoestrol. Biochem. J. 42:258. Teague, R. S. and A. E. Brown. 1951. Determination of diethylstilbestroI and its glucuronide in urine..l. Biol. Chem. 159:343.
